We compared the in vivo insulin signaling response in bone tissue of 10- verses 73-week old C57BL/6J male mice (Figure 1A). Saline or insulin (2.5 mU/kg) was interperitoneally injected and mice sacrificed after 20 min. Tibia bone was rapidly dissected and flushed with ice-cold PBS within 30 sec to remove marrow and quench phosphorylation before being snap frozen in liquid nitrogen. Protein was extracted from powdered bone tissue and digested with trypsin and LysC followed by multiplexed stable isotope labelling with Tandem Mass Tags (TMT). Enriched phosphopeptides were analyzed by multi-dimensional ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (2DLC-MS/MS). In parallel, we also quantified non-enriched peptides to analyze relative proteome abundance differences. All mice were kept on a standard chow diet.